1. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Integrating In Vitro and In Silico Methods to Evaluate Variability Barbara A. Wetmore The Hamner Institutes for Health Sciences Research Triangle Park, NC USA 27709 bwetmore@thehamner.org

2. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Broad-Based Movement in Toxicology Towards In Vitro Testing and Hazard Prediction

3. 2015 NAS Interindividual Variability Workshop | September 30, 2015 HT Toxicity Testing Data Difficulty Translating Nominal Testing Concentrations into In Vivo Doses Knudsen et al. Toxicology 282:1-15, 2011

4. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Prioritization and Hazard Prediction Based on Nominal Concentrations Can Misrepresent Potential Health Risks Protein Binding Metabolic Clearance Bioavailability van de Waterbeemd and Gifford, Nat Rev Drug Disc 2:192, 2003 Reif et al. Environ Hlth Perspect 118:1714, 2010

5. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Incorporating Dosimetry with High-Throughput Screening Data Human Hepatocytes (10 donor pool) Human Plasma (6 donor pool) Hepatic Clearance Plasma Protein Binding Population-Based In Vitro-to-In Vivo Extrapolation Population-Based In Vitro-to-In Vivo Extrapolation Plasma Concentration at Steady State for 10,000 Healthy Individuals of Both Sexes from 20 to 50 Yrs Old Setting the Stage…

6. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Using Reverse Dosimetry to Estimate Population-Based Oral Equivalent Doses Upper 95 th Percentile Css Among 10,000 Healthy Individuals of Both Sexes from 20 to 50 Yrs Old Plasma Concentration ToxCast AC 50 Value Assay X (e.g., ACE inhibition) Oral Exposure Oral Dose Required to Achieve Steady State Plasma Concentrations Equivalent to AC 50 Oral Equivalent (mg/kg/day) ToxCast AC 50 (uM) 1 mg/kg/day Upper 95 th Percentile Css (uM) = 1 mg/kg/day Oral Exposure Metabolic and binding parameters Setting the Stage…

7. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Incorporating Dosimetry and Exposure with HTS Data

8. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Primary Hepatocytes Hepatic Clearance Cl in vitro Plasma C ss General Population ≠ Plasma C ss General Population Dosimetry and Exposure Strategy Limited to General Population

9. 2015 NAS Interindividual Variability Workshop | September 30, 2015 The Impact of Population Variability on Risk Assessment Clearance results for full database (27 substrates). from Ginsberg et al., 2002, Toxicol. Sci., 66, 185-200. Ratio to young adults Age (years) from Ginsberg et al., 2005, Environ. Health Persp., 113, 1243-49. Clearance differences span across multiple newborn and juvenile life stages… … as well as geriatric life stages.

10. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Percent contribution of individual CYPs to total hepatic clearance of xanthines. Ginsberg et al., 2004, J. Toxicol. Env. Health Pt. A, 67:297-329. The Impact of Population Variability on Risk Assessment Cresteil, 1998, Food Addit. Contam., 15:45-51. Ontogeny of Human Hepatic CYP Isozymes Frequency for CYP2D6 alleles classified as functional, non-functional and reduced functioning for various subpopulations. Bradford, 2002, Pharmacogenomics. 3(2):229-43. Sole reliance on pharmacokinetic data for a “generic” population could lead to a significant underestimation of risk to a susceptible lifestage or subpopulation

11. 2015 NAS Interindividual Variability Workshop | September 30, 2015 CYP1A2 CYP2D6 CYP2C8 CYP2E1 UGT1A4 CYP3A5 CYP3A4 CYP2C19 UGT1A1 CYP2C9 CYP2B6 UGT2B7 Population-based In Vitro-In Vivo Extrapolation Primary Hepatocytes Hepatic Clearance Cl in vitro Plasma C ss General Population CYP1A2 CYP… CYP3A4 UGT… Cl rCYP1A2 Cl rCYP… Cl rCYP3A4 Cl rUGT… Plasma C ss for: Neonates Northern Europeans Asians Children And so on… Intrinsic Clearance Rates rCYP1A2 rCYP3A4 rCYP… rUGT …

12. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Integrating High-Throughput Pharmacokinetics with the ToxCast In Vitro Assays Reverse Dosimetry Oral Exposure Plasma Concentration ToxCast AC 50 Value Oral Dose Required for Specific Subpopulations to Achieve Steady State Plasma Concentrations Equivalent to In Vitro Bioactivity ~600 In Vitro ToxCast Assays Oral Equivalent Dose (mg/kg/day) What are humans exposed to? ? ? ? Chemical In Vitro Bioactivity Recombinant Enzyme Metabolism Human Plasma Protein Binding Population-Based IVIVE Model Steady State Plasma Concentrations for Different Lifestages or Subpopulations Least Sensitive Assay Most Sensitive Assay Wetmore et al., 2014, Toxicol Sci, 142(1):210-214

13. 2015 NAS Interindividual Variability Workshop | September 30, 2015 No UGT metabolism detected Carbaryl Difenoconazole Recombinant Isozyme Clearance Rates

14. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Incorporating Recombinant Phase I and II Enzyme Data into IVIVE Modeling Cl int = intrinsic clearance ISEF = Cl int, HLM Cl int, rCYP x HLM CYP abundance HLM = human liver microsomes rCYP = recombinant CYP isoform (pmol P450 / mg protn) (dimensionless) (uL / min / mg protn) (uL / min*pmol P450) Hepatic CYP Isozyme Abundance in Healthy Adults (% of Total) Scaling rCYP Data to HLM using intersystem extrapolation factors

15. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Combining Isozyme Clearance and Abundance Data to Determine Fraction Metabolized IsozymeNo. Chemicals % fm > 5% % fm RangeChemicals with % fm > 5% CYP1A230.4 - 91.4Bensulide, Carbaryl, Fludioxonil CYP2C962.1-63.1Azoxystrobin, Bensulide, Carbaryl, Difenoconazole, Haloperidol, Tebupirimfos CYP3A471.0-80.2Acetochlor, Azoxystrobin, Bensulide, Difenoconazole, Haloperidol, Lovastatin Tebupirimfos CYP3A521.4-6.4Lovastatin, Tebupirimfos UGT1A122.6-19.3Haloperidol, Tebupirimfos UGT1A430.1-12.1Difenoconazole, Haloperidol, Lovastatin

16. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Comparison of C ss Values Derived Across Multiple Lifestages and Subpopulations Upper 95 th percentile C ss Lifestage or Subpopulation (Age (yr) or Ethnic) Lifestage or Subpopulation (Age (yr) or Ethnic) Healthy Chinese Japanese North European Diabetic Renal Insufficiency 6-20 2-6 0.5-2 0-0.5 Healthy Chinese Japanese North European Diabetic Renal Insufficiency 6-20 2-6 0.5-2 0-0.5 HK AF =11.4 HK AF =11.5

17. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Comparison of C ss Values Derived Across Multiple Lifestages and Subpopulations Lifestage or Subpopulation (Age (yr) or Ethnic) Lifestage or Subpopulation (Age (yr) or Ethnic) Healthy Chinese Japanese North European Diabetic Renal Insufficiency 6-20 2-6 0.5-2 0-0.5 Healthy Chinese Japanese North European Diabetic Renal Insufficiency 6-20 2-6 0.5-2 0-0.5 HK AF =3.5 HK AF =6.7

18. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Wetmore et al., 2014, Toxicol Sci. 142(1):210-214. Comparison of C ss Values Derived Across Multiple Lifestages and Subpopulations

19. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Agreement between In Vivo and IVIVE-derived C ss Values using Recombinant CYP-based Clearance Rates Chemical In vivo PK C ss (  M) IVIVE C ss (  M) Carbaryl0.0300.046 Haloperidol0.090-0.1260.029 Lovastatin0.004-0.0090.001

20. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Estimated Chemical-Specific Toxicokinetic Adjustment Factors ChemicalMedian C ss for Healthy Population 95 th Percentile C ss for Most Sensitive Most Sensitive Estimated HK AF % Contribution of Isozyme Differences to Average HK AF Acetochlor 0.0260.15Neonatal6.786 Azoxystrobin 0.0990.66Neonatal6.786 Bensulide 0.2410.97Neonatal4.079 Carbaryl 0.0430.49Neonatal11.487 Difenoconazole 0.2010.49 Renal Insufficiency 3.599 Fludioxonil 0.384.37Neonatal11.587 Haloperidol 0.0290.14Neonatal4.983 Lovastatin 0.0010.009Neonatal6.590 Tebupirimfos 0.1070.38 Renal Insufficiency 3.515

21. 2015 NAS Interindividual Variability Workshop | September 30, 2015 ♦ ● ▼ ■ ▲ ■ ▲ ♦ Matching Oral Equivalent Doses and Exposure Estimates for Different Populations ● ▼ ♠ ♠ Subpopulation or Lifestage (Age (yr) or Ethnic) Subpopulation or Lifestage (Age (yr) or Ethnic)

22. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Matching Oral Equivalent Doses and Exposure Estimates for Different Populations

23. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Incorporating Population TD Variability with PK Variability Abdo et al. 2015, Environ Int Sep 17; 85:147-155.

24. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Key Points First comprehensive attempt to combine physiologic and PK differences to quantitate variability anticipated between life stage-, ethnic- and disease- based populations. First effort to evaluate PK variability in a manner that could 1) identify sensitive populations and 2) replace use of default safety factors in risk assessment. Subpopulation- or lifestage- based pharmacodynamic differences will also contribute to the variable susceptibilities that may be observed following chemical exposure. Tools are emerging that allow the incorporation of pharmacokinetic with pharmacodynamic variability.

25. 2015 NAS Interindividual Variability Workshop | September 30, 2015 Acknowledgements Hamner - Institute for Chemical Safety Sciences Rusty Thomas (EPA-NCCT) John Wambaugh (EPA-NCCT) Lisa M. Almond (Simcyp) Masoud Jamei (Simcyp) External Collaborators American Chemistry Council – Long Range Initiative Funding Brittany Allen Mel Andersen Harvey Clewell Alina Efremenko Eric Healy Timothy Parker Reetu Singh Mark Sochaski Longlong Yang